Stem cell niches are responsible for life-long renewal of most epithelial surfaces throughout the body. Dysregulation of these cells is believed to result in metaplasia and cancer. To date little is known about these somatic stem cell niches or their contribution to pancreatic epithelial regeneration and cancer. Our laboratory has recently discovered a novel epithelial stem cell compartment, Pancreatic Duct Glands (PDG). These glands uniquely express Shh and TFF2 as well as other developmental genes known to reside in progenitor stem cell niches. In response to acute injury these glands are capable of asymmetric division that gives rise to differentiated daughter cells which then migrate to repopulate the main ducts. In response to chronic inflammation these cells can undergo a GI metaplasia and cystic hypertrophy which histologically and molecularly resemble side-branch IPMN, known precursors of PDAC. Evaluation of human IPMN reveals that PDG form the proliferative basilar crypt segment of these large papillary projections. This proposal is focused on characterizing the role of the PDG compartment in epithelial regeneration and carcinogenesis. [SA1] The experiments proposed in this aim will determine the role of PDG as an epithelial progenitor stem cell niche responsible for epithelial renewal and regeneration. We propose to use a fate mapping, in vivo lineage tagging strategy specifically tagging the PDG compartment, in combination with a well-characterized model of acute and chronic pancreatic injury, to confirm that these compartments contain epithelial progenitor cells. In vitro and in viv strategies are also proposed to determine if these cells have multipotential capacity. Our preliminary data suggests that this compartment has at least two populations of stem cells, label retaining cells (LRC) and transient amplifying cells (TA). In order to better characterize this two stem cell population we propose a developmental sub-aim to evaluate the transcriptome by mRNA-Seq from minimal (10-cell) samples in order to identify unique markers for these cells, as well as understand key pathways that regulate this compartment during regeneration and inflammatory metaplasia. [SA2] To determine the mechanisms by which PDG contribute to the formation of pancreatic cancer precursor lesions, side-branch IPMN and cancer. To accomplish this aim we propose to use in an in vivo lineage tagging and PDG cell-specific oncogene activation strategy in mice to identify PDG as the compartment of origin for IPMN and invasive cancer. Further insight into the mechanisms underlying the formation of precursor lesions will be gained in human and mouse specimens by using a mitochondrial mutational mapping strategy to show that IPMN are formed by niche succession, monoclonal conversion and gland fission. mRNA SMART- Seq of low, moderate and high grade dysplastic IPMN may allow us to understand key pathways involved in initiation, progression, and invasion. Understanding how this compartment contributes to regeneration and cancer will give us new insights into cancer prevention, early diagnosis and treatment.